In eukaryotic cells the orderly packaging of DNA in the nucleus plays an important role in the regulation of gene transcription. Nuclear DNA is ordered in a compact complex called chromatin. The core of the complex is an octamer of highly conserved basic proteins called histones. Two each of histones H2A, H2B, H3 and H4 associate and DNA winds around the basic amino acids of the histones interacting with the negatively charged phosphate groups of the DNA. One molecule of histone H1 is associated with each wound core which accommodates approximately 146 bp of DNA. The cores are, in turn, packaged into a compact regular structure with about 200 bp of DNA between each core.
The amino terminal tails of the histones are subject to post-translational modification, in particular by acetylation of lysine. Histone deacetylases (HDACs) and histone acetyl transferases (HATs) determine the pattern of histone acetylation, which together with other dynamic sequential post-translational modifications might represent a ‘code’ that can be recognised by non-histone proteins forming complexes involved in the regulation of gene expression. This and the ability of histone deacetylases (HDACs) to also modify non-histonic substrates and participate in multi-protein complexes contributes to the regulation of gene transcription, cell cycle progression and differentiation, genome stability and stress responses. HDAC inhibitors cause the induction of differentiation, growth arrest and/or apoptosis in a broad spectrum of transformed cells in culture and tumours in animals, including both haematological cancers and solid tumours. These inhibitory effects are believed to be caused, in part, by accumulation of acetylated proteins, such as nucleosomal histones, which appear to play a major role in regulation of gene transcription. It has been recently discovered that these compounds might represent novel therapeutic agents for the treatment of genetic disorders such as β-thalassemia and sickle cell anemia.
Hemoglobin (Hb) is a tetramer of two α-like and two β-like globin polypeptide chains. In human, the genes for α-globins are clustered on chromosome 16, which contains one gene for ζ and two genes for α (α1 and α2, the proteins of which are identical). The genes for the β-like globins are clustered on chromosome 11, which contains genes for ε, β and δ, one gene for each, and two slightly different genes for γ. In addition, these clusters contain various sites that are responsible for the regulation of the expression of each gene (Steinberg, M H et al, Genetics, Pathophysiology and Clinical Management, Cambridge University Press, Cambridge, UK, 2001).
The expression of the globin genes is regulated during ontogeny. In humans, globin production is characterized by two major “switches” (Thein, S L Br. J. Haematol., 2004, 124, 264). Production of embryonic Hbs switches after the first two months into fetal Hb (HbF) (α2γ2), and then, again, before and immediately after birth, into adult Hb (HbA) (α2β2). Since both HbA and HbF contain a chains, the switch from the former to the latter represents a decrease in the expression of the γ-globin genes, associated with an increase of β-globin gene expression. The prevalence of HbF during embryonic life is explained by its high affinity to oxygen, a property that allows it to remove oxygen from HbA in the maternal red blood cells (RBCs) through the placenta.
Immediately after birth the newborn has 85-98% HbF, which gradually decreases to <5% at the age of one year. In adult life HbA is the major Hb, a small <5% is HbA2 (α2δ2) and the rest (<5%) is HbF which is concentrated in a few RBC.
β-Thalassemia and sickle cell anemia are two of the most common single gene disorders of humans. Both diseases result from different mutations of the β-globin gene that encodes two of the tetramaeric globin chains that make up the major hemoglobin present in adult red cells (adult haemoglobin, HbA).
In β-thalassemias, mutations affecting the β-globin gene or its regulatory regions cause absence (β0) or reduced (β+) synthesis of β-globin chains. This is associated with a corresponding excess of the complementary α-globin. The outcome of this unbalanced globin production is the destruction by apoptosis of erythroid precursors in the bone marrow and at the extramedullary sites (ineffective erythropoiesis) and short survival of RBCs in the peripheral blood (Bank, A. Blood, 2006, 107, 435; Stamatoyannopoulos, G. Exp. Hematol. 2005, 33, 259).
Sickle cell anemia results from a missense mutation (glutamine to valine substitution) at the 6th aminoacid of the β-globin chain. The resulting sickle Hb (HbS) forms insoluble polymers within the cytosol upon deoxygenation, with subsequent deformation of the red blood cells and vaso occlusion.
Patients with β-thalassemia and sickle cell disease do not have clinical complications of their disease at birth when their red cells contain the fetal form of Hb (HbF).
The proportion of HbF in postnatal life is influenced by various physiological and genetic factors. Epidemiological findings have shown that increased HbF in β-thalassemia ameliorates the clinical symptoms (Olivieri, N F Semin. Hematol. 1996, 33, 24; Rochette, J et al Blood Rev. 1994, 8, 213). The most convincing finding was found in individuals with mutations associated with hereditary persistence of HbF (HPFH) in adult life (Bhardwaj, U et al Mol. Diagn. 2005, 9, 151). Coexistence of homozygous β-thalassemia with HPFH is asymptomatic. It seems that HbF can functionally compensate for the absence of β-globin chains (Witt, O Am. J. Hematol. 2000, 64, 319).
In sickle cell disease, the presence of HbF reduces the effective concentration of HbS, thus decreasing the propensity for intracellular polymerization. The fetal γ-globin chains also interfere with the ability of HbS to polymerize by heterohybrid formation.
These findings have generated considerable interest in identifying molecular and pharmacological ways to increase the production of HbF. Indeed, several groups of the compounds were found to reactivate the γ-globin genes in post-natal erythroid cells.
Several findings suggest that inhibition of the activity of histone deacetylases (HDACs) is associated with an increased expression of the γ-globin genes (Cao, H Hematology, 2004, 9, 223). Among HDAC inhibitors, trichostatin was found to possess high HbF-inducing activity in human and mouse erythroleukemia cells. Witt et al (Blood, 2003, 101, 2001) showed that, among several specific HDAC inhibitors tested, apicidin was by far the most efficient HbF-inducer (at nM to μM concentrations in K562 cells) and that its effect involved, in addition to HDAC inhibition, p38 mitogen-activated protein (MAP) kinase signaling. Further HDAC inhibitors were recently characterized for their effect on human γ-globin gene expression in transgenic mice. Among the hydroxamic acid derivatives of short chain fatty acids studied butyryl and propionyl hydroxamate were most effective, increasing the human γ/murine α-globin mRNA ratios by 33.9% and 71%, respectively. This was associated with an increase in reticulocytes hematocrit, and the in vivo levels of BFU-E (Cao, H Exp. Hematol. 2005, 33, 1443).
WO 2006/061638 discloses inhibitors of histone deacetylase structurally related to the compounds of the present invention. Such compounds are useful for treating cellular proliferative diseases, including cancer, neurodegenerative disease, schizophrenia and stroke. None of the compounds disclosed in WO 2006/061638 are within the instant invention.
WO 2011/072086 discloses methods and low dose regimens for increasing fetal haemoglobin levels in patients with red blood cell disorders, by administering 2,2-dimethylbutyrate (DMB) alone or in combination with hydroxyurea, decitabine or an HDAC inhibitor.
WO 2009/141658 relates to depsipeptides which act as inhibitors of histone deacetylase (HDAC) and therefore have therapeutic utility in the treatment of conditions mediated by HDAC, including haemoglobinopathy, thalassemia and sickle cell disease.
WO 2003/087057 relates to benzamide derivatives having HDAC inhibitory activity and accordingly having potential value in the treatment of disease states associated with cancer, cystic fibrosis, Huntingtons chorea and sickle cell anemia.
However, many of these drugs have low efficacy and specificity, while some are potentially toxic or carcinogenic. There is therefore an urgent need for new agents that can induce HbF production at doses that are well tolerated by patients, thus with greater efficacy and lower toxicity with respect to known HDAC inhibitors.
The compounds of the present invention are small molecules endowed with potent HDAC inhibitory activity, capable of inducing erythroid differentiation and human γ-globin gene expression. Further, compounds of the invention are characterized by good pharmacokinetic properties in preclinical species, which are predictive of an improved therapeutic window in humans.